Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri.

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Exploring the impact of competitive compounds and catalyst synthesis method in DBT oxidative desulfurization using MoO3-V2O5/Al2O3 catalyst.

This research endeavors to address the pressing challenge of reducing sulfur content in fuels, an environmental imperative. It does so by employing bimetallic catalysts to enhance the efficiency of oxidative desulfurization (ODS) processes. This involves utilizing successive impregnation and co-impregnation methods to prepare a MoO3-V2O5/Al2O3. The catalysts underwent characterization using various techniques including X-ray diffraction (XRD), N2 adsorption-desorption, UV-vis (DRS), temperature-programmed desorption (NH3-TPD), Raman, Fourier-transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), and energy dispersive spectrum (EDS). The catalyst was utilized for the evaluation of the ODS process of dibenzothiophene (DBT). The effects of oxidants, namely H2O2 and t-butyl hydroperoxide (TBHP), were studied in the ODS. The catalyst prepared using the co-impregnation method (5M-15V-co) demonstrated significant acidic sites and exhibited remarkable efficiency in oxidative desulfurization. Remarkably, this catalyst achieved 100% oxidation of sulfur components within 30 min (min). To assess the catalyst's performance further, competitive compounds including nitrogen-containing compounds (NCCs) and saturated and unsaturated hydrocarbon compounds (HCs) were employed in the ODS. Initially, the introduction of NCCs led to a decrease in the sulfur removal rate; however, the catalyst successfully oxidized DBT completely within 60 min. When cyclohexene was present as an olefinic hydrocarbon compound, the catalyst oxidized DBT by approximately 75%, whereas DBT oxidation reached 100% within 20 min when p-xylene was introduced to the catalytic reactor. Additionally, as the O/S ratio increased from 2/5 to 10, the sulfur removal rate improved from 30 to 90%, indicating that HCs and NCCs compete with sulfur in terms of oxidant consumption.

Pt-Cu@Bi2MoO6/TiO2 Photocatalyst for CO2 Reduction.

Bi2MoO6/TiO2 heterojunction photocatalysts were constructed by depositing Bi2MoO6 nanosheets on TiO2 nanobelts' surface using a solvothermal method, and the surface of the optimum Bi2MoO6/TiO2 composite was decorated with copper and/or platinum nanoparticles. The synthesized samples were investigated for the CO2 photocatalytic reduction. The structural and optical properties of synthesized photocatalysts were characterized by XRD, FESEM, EDX, N2-physisorption, Raman, TPD-CO2, DRS, and PL analysis. The Bi2MoO6/TiO2 composite with different molar ratios of Bi2MoO6 to TiO2 (1, 1/2, 1/3, 1/4, 1/5, and 1/6) showed enhanced photocatalytic activity compared to pure Bi2MoO6 and TiO2. In comparison to bulk Bi2MoO6 and TiO2, the formation of a heterojunction between Bi2MoO6 and TiO2 leads to enhanced CO2 adsorption capacity. The enhanced performance of composites can be ascribed to the improved efficiency of light harvesting in the visible light range and suppressing charge recombination. The composite photocatalytic activity indicated that the ratio of Bi2MoO6 to TiO2 in the composite samples influenced the photocatalytic performance. The Bi2MoO6/TiO2 composite with 1/4 molar ratio had the best performance in 8 h (36.4 µmol/gcat), which was about 10 and 3 times higher than TiO2 and Bi2MoO6 photocatalysts, respectively. Under UV-visible light irradiation, the Pt-Cu@BMT4 sample produced the highest amount of methane (83.6 µmol/gcat) during CO2 photoreduction. During four irradiation cycles, the Pt-Cu@BMT4 sample exhibited superior stability with less than 5% decrease in methane production.

Different Effects of Low-Level Laser Therapy on the Proliferation of HT29 Cells in Culture and Xenograft Models.

Introduction: Different kinds of treatments have been developed to fight cancers. Low-level laser therapy (LLLT), also known as photobiomodulation therapy (PBMT), is a low-power monochromatic and coherent light that has been used successfully for healing injuries and combating malignancies. However, there are concerns about the application of LLLT to cancers due to the increased proliferation of some cancer cells after LLLT. Methods: This study investigated the effects of 650 nm and 870 nm lasers on the proliferation of HT29 colorectal cancer cell lines in vitro and in vivo. Results: The results showed that the laser with a wavelength of 870 nm did not meaningfully alter the proliferation of cultured cells. However, cell proliferation was promoted when the laser was applied within a wavelength of 650 nm. Treatment of HT29-derived tumors in nude mice with the 650 nm laser resulted in the decline of the tumor progression rate compared to controls. This result was inconsistent with the proliferative effects of the laser on the cultured cells. Conclusion: Cell behavior in response to LLLT might be different between cell culture and xenograft models.

Effect of Salicylic Acid and Pre-Cold Treatment on Flower Induction in Saffron.

Saffron is an important flowering plant, generally known as a golden condiment. The present study was performed to find the influence of different levels of SA and pre-cold treatment in the dormancy period of saffron and their effects on content enzyme activity. The results indicated that the SA2%, SA1%, and, pre-cold treatments took the shortest day to flowering. PAL enzyme activity was highest in pre-cold treatment. The higher total amount of protein was measured in the control, SA1% and SA2%. The highest amount of starch content and total soluble sugar was detected in pre-cold, SA2%, and control, respectively. No significant differences between treatments were present for CAT, PPO, GPX, and APX enzymes activity. There was a negative significant correlation between flowering time and some studied traits, i.e., starch and PAL activity. Applying SA and pre-cold treatment can induce saffron flowering and effect on pal enzyme activity and corm total protein, sugar, and starch content accordingly.

Transient expression of an scFvG8 antibody in plants and characterization of its effects on the virulence factor pthA of Xanthomonas citri subsp. citri.

Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major disease of citrus plants, causing a significant loss in the citrus industry. The pthA is a bacterial effector protein mediates protein-protein and protein-DNA interactions and modulates host transcription. Injection of pthA effector protein into the host cell induces the expression of the susceptibility gene CsLOB1 which is required for citrus canker disease development. In this study, we described in planta expression of a specific anti-pthA single-chain variable fragment (scFv) recombinant antibody, scFvG8, and assessed its function using molecular docking, immunoblotting, and indirect enzyme-linked immunosorbent assay (ELISA). Based on the results, homology-based molecular docking suggested that at least eight intermolecular hydrogen bonds are involved in pthA-scFvG8 interactions. Immunoblotting and indirect ELISA results reconfirmed specific binding of scFvG8 to pthA protein. Moreover, gene fragment encoding scFvG8 was cloned into plant expression vector and transiently expressed in leaves of Nicotiana tabacum cv. Samson by agroinfiltration method. Transient expression of scFvG8 (at the expected size of 35 kDa) in N. tabacum leaves was confirmed by western blotting. Also, immunoblotting and indirect ELISA showed that the plant-derived scFvG8 had similar activity to purified scFvG8 antibody in detecting pthA. Additionally, in scFvG8-expressing tobacco leaves challenged with Xcc, a reduction (for up to 70%) of hypersensitive response (HR) possibly via direct interaction with pthA, was observed in the necrotic leaf area compared to control plants infected with empty vector. The results obtained in this study confirm that scFvG8 can suppress the function of pthA effector protein within plant cells, thus the induction of stable expression of scFvG8 in lime trees can be considered as an appropriate approach to confer resistance to Xcc.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1.

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.

CRISPR- mediated Mutation in Cinnamoyl- CoA Reductase 4 in Allohexaploid Oilseed Crop Camelina sativa, Revealed its Pivotal Role in Resistance Against Sclerotinia sclerotiorum.

Background: Sclerotinia sclerotiorum (Ss) is a broad host range necrotrophic ascomycete fungus affecting over 400 plant species. Ss causes stem rot disease on Camelina sativa (Cs) an allohexaploid crucifer species that is promoted as a low input crop and industrial oil attributes suitable as biofuel and lubricant. Histochemical and molecular studies has linked resistance to Ss in C. sativa with the cell wall lignification (Eynck et al., 2012) and reported constitutive expression of Cinnamoyl-CoA Reductase 4 (CsCCR4) gene, in the Cs resistant line CN114263. Modern breeding efforts, such as gene editing, are needed to improve commercial lines and to limit the risk of crop loss which would be substantial to producers. Objectives: To investigate the importance of monolignol biosynthesis and the role of CsCCR4 in Camelina resistance to Ss we generated CsCCR4 knockout mutants of CN114263 Camelina line using CRISPR/Cas9-mediated gene editing. Materials and Methods: Thirty T1 plants were produced via floral dip transformation followed by glyphosate spraying that was used in the first step of screening procedures and were confirmed by PCR method. Transgene's T-DNA copy number variation, T-DNA CNV, in T1 and T2 progenitors were determined using digital droplet PCR (ddPCR) and the occurrence of mutation in the three copies of CsCCR4 homeologues in T1 and T2 generations were scrutinized by drop-off assay technique. To make sure that if the created mutants in T2 plants are real, TOPO TA sequencing flanking the Cas9/gRNA specific hot point of cleavage for three of them was conducted. Results: In the T1 generation, 25 plants were confirmed which had between one to nine T-DNA copies in the corresponding Camelina genome. In T2 generation the population were screened for potential mutation in CsCCR4 gene. Various types of mutations, including insertions and deletions, were demonstrated in three copies of CsCCR4. In fact, CRISPR system could have cut one, two or three copies of the gene in events numbered T2-plant 10, T2-plant 15 and T2-plant 19, respectively. The T3-plant 19 which showed mutation in all versions of CsCCR4 in previous generation had susceptibility to S. sclerotiorum invasion and was kept as real csccr4 mutant material for further investigations of Camelina-Sclerotinia interaction. Mutation in CsCCR4 had occurred through error-prone none- homologous end joining (NHEJ) nucleus DNA repair pathway. Ss challenge on the early flowering T3 generation. The T3 plants with mutation causing premature stop codon at position 217 of CsCCR4 were compromised in their resistance to Ss compared to the wildtype resistant control parent CN114263. Conclusion: Using ddPCR it easily was possible to identify both the T-DNA CNV and occurrence of mutation in CsCCR4 homeologues in T1 and T2 progenitors. We illustrated that CRISPR/Cas9-mediated mutation is a decent technique that can be utilized to expedite the mutant line development which could assist to figure out the activity of a CsCCR4 gene in defense responses to the pathogens in C. sativa as prospective oilseed crop for biodiesel production.

A multimodal deep learning-based drug repurposing approach for treatment of COVID-19.

Recently, various computational methods have been proposed to find new therapeutic applications of the existing drugs. The Multimodal Restricted Boltzmann Machine approach (MM-RBM), which has the capability to connect the information about the multiple modalities, can be applied to the problem of drug repurposing. The present study utilized MM-RBM to combine two types of data, including the chemical structures data of small molecules and differentially expressed genes as well as small molecules perturbations. In the proposed method, two separate RBMs were applied to find out the features and the specific probability distribution of each datum (modality). Besides, RBM was used to integrate the discovered features, resulting in the identification of the probability distribution of the combined data. The results demonstrated the significance of the clusters acquired by our model. These clusters were used to discover the medicines which were remarkably similar to the proposed medications to treat COVID-19. Moreover, the chemical structures of some small molecules as well as dysregulated genes' effect led us to suggest using these molecules to treat COVID-19. The results also showed that the proposed method might prove useful in detecting the highly promising remedies for COVID-19 with minimum side effects. All the source codes are accessible using https://github.com/LBBSoft/Multimodal-Drug-Repurposing.git.

Distinguishing drug/non-drug-like small molecules in drug discovery using deep belief network.

The advent of computational methods for efficient prediction of the druglikeness of small molecules and their ever-burgeoning applications in the fields of medicinal chemistry and drug industries have been a profound scientific development, since only a few amounts of the small molecule libraries were identified as approvable drugs. In this study, a deep belief network was utilized to construct a druglikeness classification model. For this purpose, small molecules and approved drugs from the ZINC database were selected for the unsupervised pre-training step and supervised training step. Various binary fingerprints such as Macc 166 bit, PubChem 881 bit, and Morgan 2048 bit as data features were investigated. The report revealed that using an unsupervised pre-training phase can lead to a good performance model and generalizability capability. Accuracy, precision, and recall of the model for Macc features were 97%, 96%, and 99%, respectively. For more consideration about the generalizability of the model, the external data by expression and investigational drugs in drug banks as drug data and randomly selected data from the ZINC database as non-drug were created. The results confirmed the good performance and generalizability capability of the model. Also, the outcomes depicted that a large proportion of misclassified non-drug small molecules ascertain the bioavailability conditions and could be investigated as a drug in the future. Furthermore, our model attempted to tap potential opportunities as a drug filter in drug discovery.

Bacterial Secretome Analysis in Hunt for Novel Bacteriocins with Ability to Control Xanthomonas citri subsp. Citri.

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causative agent of bacterial citrus canker, has affected citriculture worldwide. Varieties of means have been used to minimize its devastating effects, but no attention has been given to bacteriocins. OBJECTIVES: Here and for the first time, we report the isolation and characterization of two novel bacteriocins. MATERIALS AND METHODS: Secretome containing bacteriocins of isolated bacteria was separated via SDS-PAGE. Each isolated protein band was characterized and checked for its efficacy in controlling two pathogenic isolates of Xcc via disk diffusion assay. The effects of varieties of carbon, nitrogen and phosphate sources were evaluated on both bacterial growth and bacteriocin production via Taguchi orthogonal method. RESULTS: The two bacteriocins showed an activity up to 55ºC that were sensitive to proteases suggesting being protein in nature. Analysis of SDS-PAGE purified protein bands of bacterial secretomes with demonstrated potency against Xcc revealed the presence of peptides with relative molecular masses of 16.9 and 17 kDa for Cronobacter and Enterobacter, respectively. Sequence analysis of peptides revealed an HCP1 family VI secretion system homologue for Cronobacter (YP_001439956) and pilin FimA homologue for Enterobacter (CBK85798.1). A Taguchi orthogonal array was also implemented to determine the effect of temperature and eight other chemical factors on bacteriocin production for each bacterium. CONCLUSIONS: Two peptides with novel antibacterial activities effective against Xcc were isolated, characterized and conditions were optimized for their higher production.

Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68.

The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68

The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

Lactose consuming strains of Xanthomonas citri subsp. citri (Xcc) insight into the emergence of natural field resources for xanthan gum production.

Xanthomonas genus possesses a low level of ß-galactosidase gene expression and is therefore unable to produce xanthan gum in lactose-based media. In this study, we report the emergence of some natural field strains of Xanthomonas citri subsp. citri (Xcc) capable to use lactose as a sole carbon source to produce xanthan gum. From 210 Xcc strains isolated from key lime (C. aurantifolia), 27 showed the capacity to grow on lactose containing medium. Xcc lactose consuming strains demonstrated a good level of xanthan production. Amongst all, NIGEBK37 produced the greatest (14.62 g/l) amount of xanthan gum in experimental laboratory conditions. By evaluating the viscosity of the biopolymer at 25 °C, it was demonstrated that xanthan synthesized by strain NIGEBK37 has the highest viscosity (44,170.66 cP). Our results were indicative for the weakness of a commercial strain of Xanthomonas campestris pv. Campestris DSM1706 (Xcc/DSM1706) to produce xanthan in lactose containing medium.

Role of symbiotic bacteria in the growth and development of the Sunn pest, Eurygaster integriceps.

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is the most important pest of wheat and barley in wide areas of the world. Different aspects of the insect's life history have been studied, but to date nothing is known about their microbial symbionts. Here, the contribution of symbiotic bacteria to the fitness of the bug was investigated by combining two different approaches to manipulate the host's microbial community: the supplementation of antibiotics into the insects' diet and egg surface sterilization. First, bacteria cultured from gut homogenates were subjected to antibiotic screening tests using 20 different antibiotics. Norfloxacin was the most effective antibiotic, with the greatest inhibition zone among all antibiotics tested. Feeding norfloxacin to adult E. integriceps individuals significantly impaired growth and development of the offspring in a dose-dependent manner, i.e., higher antibiotic doses increased the negative effects on nymphal growth and development. Total developmental time from first nymphal instars to adult emergence in control animals was 30.1 days, but when adults had been offered diets with 10, 20, and 30 µg antibiotic per mg diet, the offspring's developmental time was prolonged to 32.8, 34.0, and 34.8 days, respectively. In the highest two doses of norfloxacin, all of the nymphs died before reaching the fifth nymphal instar. Similar results as for the antibiotic treatment were obtained when egg surface sterilization was used to manipulate the microbial community of E. integriceps. These results indicate that bacterial symbionts play a crucial role in the successful development of the host.

Insect inducible antimicrobial peptides and their applications.

Antimicrobial peptides (AMPs) are found as important components of the innate immune system (host defense) of all invertebrates. These peptides can be constitutively expressed or induced in response to microbial infections. Indeed, they vary in their amino acid sequences, potency and antimicrobial activity spectra. The smaller AMPs act greatly by disrupting the structure or function of microbial cell membranes. Here, the insect innate immune system with emphasis on inducible antimicrobial peptide properties against microbial invaders has been discussed.

Assessment of the genetic diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a basis to identify putative pathogenicity genes and a type III secretion system of the SPI-1 family by multiple suppression subtractive hybridizations.

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.

Characterization of ISXax1, a novel insertion sequence restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and Xanthomonas axonopodis pv. vesicatoria.

ISXax1 is a novel insertion sequence belonging to the IS256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that ISXax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with ISXax1 insertion in these strains.